Biology subjects

Le Saux, T.

Publications and source records attributed to Le Saux, T..

5 recordsLinked to original sources

bioRxiv2026

Non-covalent reversibly photoconvertible fluorescent tags for wash-free protein labeling

Reversibly photoswitchable fluorophores are widely used in advanced bioimaging but their design remains demanding. Here, we introduce a new series spanning the whole visible range, which results from combining a large set of fluorogens with the FAST protein scaffold. We first demonstrate that these well-established labeling fluorescent protein tags turn into negative reversible photoswitchers upon decreasing the fluorogen concentration and increasing light intensity. We then show that using not anymore one but two fluorogens adds new responses to illumination. Thus, we obtain positive reversible photoswitchers, that increase their brightness under illumination. We also generate a palette of non-covalent reversibly photoconvertible fluorescent proteins changing their fluorescence color upon illumination, a reversible behavior that still remains absent in regular fluorescent proteins. This light-induced color change opens the possibility to discriminate six spectrally similar FAST variants in live cells upon demonstrating the superiority of using multiple spectral channels for exploiting the time dependence of the fluorescence response to illumination.

biophysics↗

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

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↗

bioRxiv2019

Design of a comprehensive microfluidic and microscopic toolbox for the ultra-wide spatio-temporal study of plant protoplasts development and physiology

BackgroundOne of the main features of plant cells is their strong plasticity, and their propensity to regenerate an organism from a single cell. Plant protoplasts are basic plant cells units in which the pecto-cellulosic cell wall has been removed, but the plasma membrane is intact. One of the main features of plant cells is their strong plasticity, which in some species, can be very close from what is defined as cell totipotency. Methods and differentiation protocols used in plant physiology and plant biology usually involve macroscopic vessels and containers that make difficult, for example, to follow the fate of the same protoplast all along its full development cycle, but also to perform continuous studies of the influence of various gradients in this context. These limits have hampered the precise study of regeneration processes. ResultsHerein, we present the design of a comprehensive, physiologically relevant, easy-to-use and low-cost microfluidic and microscopic setup for the monitoring of Physcomitrella patens (P. patens) growth and development on a long-term basis. The experimental solution we developed is made of two parts (i) a microfluidic chip composed of a single layer of about a hundred flow-through microfluidic traps for the immobilization of protoplasts, and (ii) a low-cost, light-controlled, custom-made microscope allowing the continuous recording of the moss development in physiological conditions. We validated the experimental setup with three proofs of concepts: (i) the kinetic monitoring of first division steps and cell wall regeneration, (ii) the influence of the photoperiod on growth of the protonemata, and (iii) finally the induction of leafy buds using a phytohormone, cytokinin. ConclusionsWe developed the design of a comprehensive, physiologically relevant, easy-to-use and low-cost experimental setup for the study of P. patens development in a microfluidic environment. This setup allows imaging of P. patens development at high resolution and over long time periods.

plant biology↗