Biology subjects

Gautier, A.

Publications and source records attributed to Gautier, A..

28 records · Page 2Linked to original sources

bioRxiv2023

Improving split reporters of protein-protein interactions through orthology-based protein engineering

Protein-protein interactions (PPI) can be detected through selective complementation of split fluorescent reporters made of two complementary fragments that reassemble into a functional fluorescent reporter when in close proximity. We previously introduced splitFAST, a chemogenetic PPI reporter with rapid and reversible complementation. Here, we present the engineering of RspA-splitFAST, an improved reporter displaying higher brightness, lower self-complementation and higher dynamic range for optimal monitoring of PPI using an original protein engineering strategy that exploits proteins with orthology relationships. Our study allowed the identification of a system with improved properties and enabled a better understanding of the molecular features controlling the complementation properties. Because of the rapidity and reversibility of its complementation, its low self-complementation, high dynamic range, and improved brightness, RspA-splitFAST is well suited to study PPI with high spatial and temporal resolution, opening great prospects to decipher the role of PPI in various biological contexts.

cell biology↗

bioRxiv2023

A fluorogenic chemically induced dimerization technology for controlling, imaging and sensing protein proximity

Proximity between proteins plays an essential and ubiquitous role in many biological processes. Molecular tools enabling to control and observe the proximity of proteins are essential for studying the functional role of physical distance between two proteins. Here we present CATCHFIRE (Chemically Assisted Tethering of CHimera by Fluorogenic Induced REcognition), a chemically induced proximity technology with intrinsic fluorescence imaging and sensing capabilities. CATCHFIRE relies on genetic fusion to small dimerizing domains that interact upon addition of fluorogenic inducers of proximity that fluoresce upon formation of the ternary assembly, allowing real-time monitoring of the chemically induced proximity. CATCHFIRE is rapid and fully reversible, and allows the control and tracking of protein localization, protein trafficking, organelle transport and cellular processes, opening new avenues for studying or controlling biological processes with high spatiotemporal resolution. Its fluorogenic nature allowed furthermore the design of innovative biosensors for the study of various processes, such as signal transduction and apoptosis.

cell biology↗

bioRxiv2022

A population modification gene drive targeting both Saglin and Lipophorin disables Plasmodium transmission in Anopheles mosquitoes

Lipophorin is an essential, highly expressed lipid transport protein that is secreted and circulates in insect hemolymph. We hijacked the Anopheles coluzzii Lipophorin gene to make it co-express a single-chain version of antibody 2A10, which binds sporozoites of the malaria parasite Plasmodium falciparum. The resulting transgenic mosquitoes show a markedly decreased ability to transmit Plasmodium berghei expressing the P. falciparum circumsporozoite protein. To force the spread of this anti-malarial transgene in a mosquito population, we designed and tested several CRISPR/Cas9-based gene drives. One of these is installed in, and disrupts, the pro-parasitic gene Saglin and also cleaves wild-type Lipophorin, causing the anti-malarial modified Lipophorin version to replace the wild type and hitch-hike together with the Saglin drive. Although generating drive-resistant alleles and showing instability in its gRNA-encoding multiplex array, the Saglin-based gene drive reached high levels in caged mosquito populations and efficiently promoted the simultaneous spread of the antimalarial Lipophorin::Sc2A10 allele. This combination is expected to decrease parasite transmission via two different mechanisms. This work contributes to the design of novel strategies to spread antimalarial transgenes in mosquitoes, and illustrates some expected and unexpected outcomes encountered when establishing a population modification gene drive.

genetics↗

bioRxiv2022

The salivary protein Saglin facilitates efficient midgut colonization of Anopheles mosquitoes by malaria parasites

Malaria is caused by the unicelullar parasite Plasmodium which is transmitted to humans through the bite of infected female Anopheles mosquitoes. To initiate sexual reproduction and to infect the midgut of the mosquito, Plasmodium gametocytes are able to recognize the intestinal environment after being ingested during blood feeding. A shift in temperature, pH change and the presence of the insect-specific compound xanthurenic acid have been shown to be important stimuli perceived by gametocytes to become activated and proceed to sexual reproduction. Here we report that the salivary protein Saglin, previously proposed to be a receptor for the recognition of salivary glands by sporozoites, facilitates Plasmodium colonization of the mosquito midgut, but does not contribute to salivary gland invasion. In mosquito mutants lacking Saglin, Plasmodium infection of Anopheles females is greatly reduced, resulting in impaired transmission of sporozoites at low infection densities. Interestingly, our results suggest that Saglin, present in mosquito saliva, must be ingested with the blood meal to affect Plasmodium development, indicating a previously unknown host-pathogen interaction. Furthermore, we were able to show that saglin deletion has no fitness cost in laboratory conditions, suggesting this gene would be an interesting target for gene drive approaches.

microbiology↗

bioRxiv2021

Reciprocal regulation of Shh trafficking and H2O2 levels via a noncanonical BOC-Rac1 pathway

Among molecules that bridge environment, cell metabolism, and cell signaling, H2O2 recently appeared as an emerging but central player. Its level depends on cell metabolism and environment and was recently shown to play key roles during embryogenesis, contrasting with its long-established role in disease progression. We decided to explore whether the secreted morphogen Sonic hedgehog (Shh), known to be essential in a variety of biological processes ranging from embryonic development to adult tissue homeostasis and cancers, was part of these interactions. Here, we report that H2O2 levels control key steps of Shh delivery and that physiological in vivo modulation of H2O2 levels changes Shh distribution and tissue patterning. A feedback loop exists in which Shh trafficking controls H2O2 synthesis via a non-canonical BOC-Rac1 pathway, leading to cytoneme growth. Our findings reveal that Shh directly impacts its own distribution, thus providing a molecular explanation for the robustness of morphogenesis to both environmental insults and individual variability.

cell biology↗

bioRxiv2021

An engineered multifunctional protein tag for advanced fluorescence imaging

Biocompatible fluorescent reporters with spectral properties spanning the entire visible spectrum are indispensable tools for imaging the biochemistry of living cells and organisms in real time. Here, we present the engineering of a fluorescent chemogenetic reporter with tunable optical and spectral properties. A collection of live-cell compatible fluorogenic chromophores with various electronic properties enables to generate bimolecular fluorescent assemblies that cover the visible spectrum from blue to red using a single protein tag engineered and optimized by directed evolution and rational design. We showed that the ability to tune the fluorescence color and properties through simple molecular modulation provides an unprecedent experimental versatility for imaging proteins in live cells, including delicate cultured hippocampal neurons, and in multicellular organisms. The ability to tune the spectral properties and fluorescence performance enables to match the spectral specifications and requirements of the most advanced imaging techniques, and allowed us to achieve efficient stimulated emission depletion (STED) nanoscopy of fusion proteins in live cells and live primary cultured neurons.

molecular biology↗

bioRxiv2020

Visualizing the dynamics of exported bacterial proteins with the chemogenetic fluorescent reporter FAST

Bacterial proteins exported to the cell surface play key cellular functions. However, despite the interest to study the localization of surface proteins such as adhesins, transporters or hydrolases, monitoring their dynamics in live imaging remains challenging, due to the limited availability of fluorescent probes remaining functional after secretion. In this work, we used the Escherichia coli intimin and the Listeria monocytogenes InlB invasin as surface exposed scaffolds fused with the recently developed chemogenetic fluorescent reporter protein FAST. Using both membrane permeant (HBR-3,5DM) and non-permeant (HBRAA-3E) fluorogens that fluoresce upon binding to FAST, we demonstrated that fully functional FAST can be exposed at the cell surface and specifically tagged on the external side of the bacterial envelop in both diderm and monoderm bacteria. Our work opens new avenues to study of the organization and dynamics of the bacterial cell surface proteins.

microbiology↗

bioRxiv2020

Orthogonal fluorescent chemogenetic reporters for multicolor imaging

Fluorescence microscopy is an indispensable tool in biological research, allowing sub-second and sub-micrometer mapping of molecules or processes inside living cells. Moreover, using spectrally separated fluorophores, one can observe multiple targets simultaneously, leading to a deeper understanding of the dynamic molecular interplays that regulate cell function and fate. Chemogenetic systems, which combine a protein tag and a synthetic fluorophore, provide certain advantages over fluorescent proteins since there is no requirement for chromophore maturation. However, the fluorophore promiscuity of chemogenetic systems renders two-color applications challenging. Here, we present the engineering of a set of spectrally orthogonal fluorogen activating tags based on the Fluorescence Activating and absorption Shifting Tag (FAST), that are compatible with two-color, live cell imaging. The resulting tags, greenFAST and redFAST, demonstrate orthogonality not only in their fluorogen recognition capabilities, but also in their one- and two-photon absorption profiles. A two-color cell cycle sensor based on greenFAST and redFAST is capable of detecting very short, early cell cycles in zebrafish development which had previously been difficult to image. Furthermore, this pair of orthogonal tags can be developed into split complementation systems that are capable of detecting multiple protein-protein interactions by live cell fluorescence microscopy.

molecular biology↗

bioRxiv2020

A far-red fluorescent chemogenetic reporter for in vivo molecular imaging

Far-red emitting fluorescent labels are highly desirable for spectral multiplexing and deep tissue imaging. Here, we describe the generation of frFAST (far-red Fluorescence Activating and absorption Shifting Tag), a 14-kDa monomeric protein that forms a bright far-red fluorescent assembly with (4-hydroxy-3-methoxy-phenyl)allylidene rhodanine (HPAR-3OM). As HPAR-3OM is essentially non-fluorescent in solution and in cells, frFAST can be imaged with high contrast in presence of free HPAR-3OM, which allowed the rapid and efficient imaging of frFAST fusions in live cells, zebrafish embryo/larvae and chicken embryo. Beyond enabling genetic encoding of far-red fluorescence, frFAST allowed the design of a far-red chemogenetic reporter of protein-protein interactions, demonstrating its great potential for the design of innovative far-red emitting biosensors.

molecular biology↗

bioRxiv2019

Listeriolysin O promotes the intravacuolar growth of Listeria monocytogenes in epithelial cells

Real-time imaging of bacterial virulence factor dynamics is hampered by the limited number of fluorescent tools suitable for tagging secreted effectors. Here, we demonstrated that the fluorogenic reporter FAST could be used to tag secreted proteins, and we implemented it to monitor infection dynamics in epithelial cells exposed to the human pathogen Listeria monocytogenes (Lm). By tracking individual FAST-labelled vacuoles after Lm internalisation into cells, we unveiled the heterogeneity of residence time inside entry vacuoles. Although half of the bacterial population escaped within 13 minutes after entry, 12% of bacteria remained entrapped over an hour inside long term vacuoles, and sometimes much longer, regardless of the secretion of the pore-forming toxin listeriolysin O (LLO). We imaged LLO-FAST in these long-term vacuoles, and showed that LLO enabled Lm to proliferate inside these compartments, reminiscent of what had been previously observed for Spacious Listeria-containing phagosomes (SLAPs). Unexpectedly, inside epithelial SLAP-like vacuoles (eSLAPs), Lm proliferated as fast as in the host cytosol. eSLAPs thus constitute an alternative replication niche in epithelial cells that might promote the colonization of host tissues. Author summaryBacterial pathogens secrete virulence factors to subvert their hosts; however, monitoring bacterial secretion in real-time remains challenging. Here, we developed a convenient method that enabled fluorescent imaging of secreted proteins in live microscopy, and applied it to the human pathogen Listeria monocytogenes. Listeria has been described to invade cells and proliferate in their cytosol; it is first internalized inside vacuoles, from where it escapes thanks to the secretion of virulence factors that disrupt membranes. Our work revealed the existence, in human epithelial cells, of a population of Listeria that failed to escape vacuoles but instead multiplied efficiently therein, despite -and in fact, thanks to-- the active secretion of a toxin that permeates membranes. This intravacuolar niche may provide Listeria with an alternative strategy to colonize its host.

microbiology↗