Biology subjects

Jullien, L.

Publications and source records attributed to Jullien, L..

4 recordsLinked to original sources

bioRxiv2025

Photoejection turns non-covalent fluorescent tags into negative reversible photoswitchers

Reversibly photoswitchable fluorophores have enabled a broad range of applications in advanced fluorescence bioimaging. Here, we introduce RSpFAST, a new class of reversibly photoswitchable fluorescent labels that combine a biomolecular host (pFAST protein tag) with a reversibly photoisomerizable guest (fluorogen), allowing fluorescence brightness to be modulated through illumination and molecular complexation. We combine thermokinetic, photochemical, and structural investigations to obtain a comprehensive mechanistic and kinetic understanding of RSpFAST. Building on this theoretical framework, we demonstrate in both live and fixed cells that RSpFAST exhibits an unprecedented dual behavior: a stable and wash-free fluorescent labeling tag turns into a negative reversible photoswitcher by lowering the fluorogen concentration and increasing light intensity. In this photoejection-driven kinetic regime, RSpFAST is shown to be an efficient marker for dynamic contrast and super-resolution microscopy.

biochemistry↗

bioRxiv2025

Chemogenetic modulation of luciferase emission color for imaging and sensing

Bioluminescent luciferases have emerged as powerful tools for bioimaging, enabling to image biological systems without external excitation light, reducing thus phototoxicity and eliminating background autofluorescence. Advanced imaging requires luciferases that deliver high photon output for enhanced sensitivity, tunable emission colors for multicolor imaging, and red-shifted emission for effective deep tissue imaging. Here, we introduce LumiFAST, a small tunable luciferase engineered by fusing the bright blue-light emitting NanoLuc with the tunable chemogenetic fluorescent reporter pFAST. pFAST binds and stabilizes the fluorescent state of a variety of synthetic fluorogenic chromophores (also called fluorogens). Its proximity with NanoLuc leads to efficient bioluminescence resonance energy transfer (BRET), enabling customizable red-shifted emission. Thanks to the small size of pFAST, LumiFAST maintains a compact structure, while its modular design allows emission color to be tuned from cyan to green, yellow, orange and red simply by changing the fluorogen. Systematic optimization of the fusion topology and linker length yielded an optimal variant with apparent BRET efficiencies reaching up to 90 %. The red-shifted emission of LumiFAST enables dual-color microscopy imaging when used alongside NanoLuc and allows imaging through thick scattering media. Beyond imaging, our insights into the structural factors governing efficient BRET allowed us to engineer biosensors based on NanoLuc and pFAST for the visualization of protease activity and protein-protein interactions in live cells.

cell biology↗

bioRxiv2025

Interplay between high-energy quenching and state transitions in Chlamydomonas reinhardtii: a single-cell approach

Studying cell-to-cell heterogeneity is essential to understand how unicellular organisms respond to stresses. We introduce a single-cell analysis framework that enables the study of intercellular heterogeneity of photosynthetic traits, particularly their interactions within individual cells that have identical genotypes, cellular contexts and histories. Our approach combines single-cell imaging of chlorophyll a fluorescence with machine learning and we study light stress responses in Chlamydomonas reinhardtii as a proof-of- concept. This framework allows us to score the extent of high-light responses such as state transitions (qT) and high-energy quenching (qE), to reveal significant cell-to-cell heterogeneity and to reveal a strong correlation between qT and qE, undetectable in bulk measurements. This study highlights the value of single-cell phenotypic analysis for for investigating light stress responses in unicellular organisms. We detail the key aspects that come into play to generalize the method to other complex stress responses involving multiple traits.

biophysics↗

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↗