Biology subjects

Dedecker, P.

Publications and source records attributed to Dedecker, P..

At least 19 recordsLinked to original sources

bioRxiv2026

A genetically-encoded cysteine biosensor to monitor cysteine dynamics across life domains

Cysteine is a central metabolite in cellular redox regulation and iron-sulfur cluster assembly. Despite its critical role, monitoring cysteine dynamics in living systems has remained a challenge due to the lack of tools that avoid cysteine oxidation and/or do not destroy the cell in the process. Here, we report the development of Cystector (from Cysteine Detector), a genetically encoded, ratiometric green fluorescent biosensor for cysteine that exhibits an exceptional selectivity, minimal pH sensitivity in the physiological range, and a dynamic range of up to 4500%. Furthermore, the sensor retains functionality in the presence of physiological glutathione concentrations. We demonstrate the live-cell functionality of Cystector by monitoring intracellular and extracellular cysteine dynamics in different organisms. In E. coli, we show how cystine reduction in Escherichia coli is dependent on glutathione and glutaredoxins, and that the reduced cysteine is then exported into the extracellular environment. In yeast, we demonstrate how energy metabolism and oxidative stress determine cysteine homeostasis. In mammalian cells, we show how Cystector effectively monitors cysteine depletion in response to treatments such as H2O2, erastin2, and glutamate. Finally, we demonstrate, via a mitochondrially targeted variant, that Cystector can be used to monitor subcellular cysteine dynamics. These results together establish Cystector as a robust tool to unravel cysteine metabolism and transport in live cells across life domains.

molecular biology↗

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

NanoBlocks: creating fluorescent biosensors from affinity binders using competitive binding

Genetically-encoded fluorescent biosensors have revolutionized our understanding of complex systems by permitting the in situ observation of chemical activities. However, only a comparatively small set of chemical activities can be monitored, largely due to the need to identify protein domains that undergo conformational and/or association changes in response to a stimulus. Here, we present a strategy that can convert simple affinity binders such as nanobodies into biosensors for their innate targets by introducing a peptide sequence that competes for the binding site. We demonstrate proof-of-concept implementations of this NanoBlock design, developing sensors based on the ALFA nanobody and on the PDZ domain of Erbin. We show that these sensors can reliably detect their targets in vitro, in mammalian cells, and as part of fluorescence-activated cell sorting (FACS) experiments. In doing so, our strategy offers a way to strongly expand the range of cellular processes that can be probed using fluorescent biosensors.

biochemistry↗

bioRxiv2024

DynaMAP: Dynamic Microbiome Abundance Profiling through high density optical mapping

Microbial abundance profiling is rapidly becoming an essential method in biomedical research, though it is often costly and time intensive. We present DynaMAP, a rapid approach for microbiome profiling that uses single-molecule imaging to develop an optical map of metagenomic DNA. DynaMAP achieves strain-level taxonomic profiling without requiring base-by-base sequencing and with a one-day turnaround time. In doing so, it delivers microbiome profiling that is comparable to shotgun sequencing but operates more efficiently, strongly expanding the possibility for microbiome analysis.

microbiology↗

bioRxiv2023

Per-pixel unmixing of spectrally overlapping fluorophores using intra-exposure excitation modulation

Multilabel fluorescence imaging is essential for the visualization of complex systems, though a major challenge is the limited width of the usable spectral window. Here, we present a new method, exNEEMO, that enables per-pixel quantification of spectrally-overlapping fluorophores based on their light-induced dynamics, in a way that is compatible with a very broad range of timescales over which these dynamics may occur. Our approach makes use of intra-exposure modulation of the excitation light to distinguish the different emitters given their reference responses to this modulation. We use approach to simultaneously image four green photochromic fluorescent proteins at the full spatial resolution of the imaging. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/538742v2_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1f49247org.highwire.dtl.DTLVardef@1af85c2org.highwire.dtl.DTLVardef@768a92org.highwire.dtl.DTLVardef@83b81b_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

The TriScan: fast and sensitive 3D confocal fluorescence imaging using a simple optical design

We present the TriScan, a compact and inexpensive fluorescence microscope that can combine the speed of widefield microscopy with the 3D-sectioning capabilities of confocal microscopy. The optical layout is based on a module that combines line-scan confocal imaging with a sensitive camera detector, realized using a simple optical layout that permits the use of arbitrarily fast scanning mirrors. The resulting design is theoretically capable of full field-of-view acquisition rates in the kilohertz regime combined with a diffraction-limited resolution and single-molecule sensitivity. In doing so, the system provides the ease-of- use and speed of widefield imaging combined with the optical sectioning of one-photon confocal imaging. The simple and inexpensive design is suitable for a broad variety of settings ranging from research to diagnostics and screening.

biophysics↗

bioRxiv2023

Fast and artifact-free excitation multiplexing using synchronized image scanning

We present the Resonator, a simple optical device that provides quasi-simultaneous fluorescence imaging with multiple excitation wavelengths. The device uses a resonant scanning mirror to periodically displace the sample image on a camera sensor at a rate that is much faster than the image acquisition rate. The excitation light is synchronized with the scanner motion to create two laterally shifted copies of the image, each containing the fluorescence excited by a single wavelength. The additional information is then encoded either into the point-spread function of the imaging or as multiple distinct images. Since this multiplexing is performed at very high rates, our design can eliminate or mitigate artifacts caused by temporal aliasing in conventional sequential imaging. We demonstrate the use of our system for the monitoring of fast light-induced dynamics in single quantum dots and for the imaging of Ca2+ signalling in hippocampal neurons.

biophysics↗

bioRxiv2023

Oxygen-induced chromophore degradation in the photoswitchable red fluorescent protein rsCherry

Reversibly switchable monomeric Cherry (rsCherry) is a photoswitchable variant of the red fluorescent protein mCherry. We report that this protein gradually and irreversibly loses its red fluorescence in the dark over a period of months at 4{degrees}C and a few days at 37{degrees}C. We also find that its ancestor, mCherry, undergoes a similar fluorescence loss but at a slower rate. X-ray crystallography and mass spectrometry reveal that this is caused by the cleavage of the p-hydroxyphenyl ring from the chromophore and the formation of two novel types of cyclic structures at the remaining chromophore moiety. Overall, our work sheds light on a new process occurring within fluorescent proteins, further adding to the chemical diversity and versatility of these molecules.

biophysics↗

bioRxiv2022

Simultaneous multicolor fluorescence imaging using duplication-based PSF engineering

We present a way to encode more information in fluorescence imaging by splitting the emission into copies of the original point-spread function (PSF), which offers broadband operation and compatibility with other PSF engineering modalities and existing analysis tools. We demonstrate the approach using the Circulator, an add-on that encodes the fluorophore emission band into the PSF, enabling simultaneous multicolor super-resolution and single-molecule microscopy using essentially the full field of view.

biophysics↗

bioRxiv2021

Photochromic fluorophores enable imaging of lowly-expressed proteins in the autofluorescent fungus Candida albicans

Fluorescence microscopy is a standard research tool in many fields, though collecting reliable images can be difficult in systems characterized by low expressions levels and/or high background fluorescence. We present the combination of a photochromic fluorescent protein and stochastic optical fluctuation imaging (SOFI) to deliver suppression of the background fluorescence. This strategy makes it possible to resolve lowly- or endogenously-expressed proteins, as we demonstrate for Gcn5, a histone acetyltransferase required for complete virulence, and Erg11, the target of the azole antifungals agents in the fungal pathogen C. albicans. We expect that our method can be readily used for sensitive fluorescence measurements in systems characterized by a high background fluorescence. ImportanceUnderstanding the spatial and temporal organization of proteins-of-interest is key to unravel cellular processes and identify novel possible antifungal targets. Only a few therapeutic targets have been discovered in Candida albicans and resistance mechanisms against these therapeutic agents is rapidly acquired. Fluorescence microscopy is a valuable tool to investigate molecular processes and assess the localization of possible antifungal targets. Unfortunately, fluorescence microscopy of C. albicans suffers from extensive autofluorescence. In this work we present the use of a photochromic fluorescent protein and stochastic optical fluctuation imaging to enable imaging of lowly-expressed proteins in C. albicans through the suppression of autofluorescence. This method can be applied in C. albicans research or adapted for other fungal systems allowing the visualization of intricate processes.

cell biology↗

bioRxiv2021

Self-contained and modular structured illumination microscope

We present a modular implementation of structured illumination microscopy (SIM) that is fast, largely self-contained and that can be added onto existing fluorescence microscopes. Our instrument, which we call HIT-SIM, can theoretically deliver well over 50 super-resolved images per second and is readily compatible with existing acquisition software packages. We provide a full technical package consisting of schematics, a list of components and an alignment scheme that provides detailed specifications and assembly instructions. We illustrate the performance of the instrument by imaging optically large samples containing sequence-specifically stained DNA fragments.

biophysics↗

bioRxiv2021

Simultaneous readout of multiple FRET pairs using photochromism

Forster resonant energy transfer (FRET) is a powerful mechanism to probe associations in situ. Simultaneously performing more than one FRET measurement can be challenging due to the spectral band-width required for the donor and acceptor fluorophores. We present an approach to distinguish overlapping FRET pairs based on the photochromism of the donor fluorophores, even if the involved fluorophores display essentially identical absorption and emission spectra. We develop the theory underlying this method and validate our approach using numerical simulations. To apply our system, we develop rsAKARev, a photochromic biosensor for cAMP-dependent kinase (PKA), and combine it with the spectrally-identical biosensor EKARev, a reporter for ERK kinase activity, to deliver simultaneous readout of both activities in the same cell. We further perform multiplexed PKA, ERK, and calcium measurements by including a third, spectrally-shifted biosensor. Our work demonstrates that exploiting donor photochromism in FRET can be a powerful approach to simultaneously read out multiple associations within living cells.

biophysics↗

bioRxiv2020

Absolute measurement of cellular activities using photochromic single-fluorophore biosensors

Genetically-encoded biosensors based on a single fluorescent protein are widely used to visualize analyte levels or enzymatic activities in cells, though usually to monitor relative changes rather than absolute values. We report photochromism-enabled absolute quantification (PEAQ) biosensing, a method that leverages the photochromic properties of biosensors to provide an absolute measure of the analyte concentration or activity. We develop proof-of-concept photochromic variants of the popular GCaMP family of Ca2+ biosensors, and show that these can be used to resolve dynamic changes in the absolute Ca2+ concentration in live cells. We also show how our method can be expanded to fast imaging with reduced illumination intensities or to situations where the absolute illumination intensities are unknown. In principle, PEAQ biosensing can be applied to other biosensors with photochromic properties, thereby expanding the possibilities for fully quantitative measurements in complex and dynamic systems.

biophysics↗

bioRxiv2020

Integrated structure-function dataset reveals key mechanisms underlying photochromic fluorescent proteins

Photochromic fluorescent proteins have become versatile tools in the life sciences, though our understanding of their structure-function relation is limited. Starting from a single scaffold, we have developed a range of 27 photochromic fluorescent proteins that cover a broad range of spectroscopic properties, yet differ only in one or two mutations. We also determined 43 different crystal structures of these mutants. Correlation and principal component analysis of the spectroscopic and structural properties confirmed the complex relationship between structure and spectroscopy, suggesting that the observed variability does not arise from a limited number of mechanisms, but also allowed us to identify consistent trends and to relate these to the spatial organization around the chromophore. We find that particular changes in spectroscopic properties can come about through multiple different underlying mechanisms, of which the polarity of the chromophore environment and hydrogen bonding of the chromophore are key modulators. Furthermore, some spectroscopic parameters, such as the photochromism, appear to be largely determined by a single or a few structural properties, while other parameters, such as the absorption maximum, do not allow a clear identification of a single cause. We also highlight the role of water molecules close to the chromophore in influencing photochromism. We anticipate that our dataset can open opportunities for the development and evaluation of new and existing protein engineering methods.

biophysics↗

bioRxiv2020

Active focus stabilization for fluorescence microscopy by tracking actively generated infrared spots.

Fluorescent time-lapse experiments often suffer from focus drift, regularly rendering long measurements partially unusable. Frequently, this instability can be traced back to the specific mechanical components of the setup, but even in highly robust implementations z-drift occurs due to small temperature fluctuations which are hard to avoid. To resolve this issue, microscope manufacturers often offer their own interpretation of out-of-focus correction modules for their flagship instruments. However, self-assembled or older systems typically have to fend for their own or adapt their measurements to circumvent drift effects. In this manuscript, we propose a cost-efficient z-drift detection- and correction system that, due to its modular design, can be attached to any fluorescence microscope with an actuated stage or objective, be it in a custom or commercial setup. The reason for this wide applicability is specific to the design, which has a straightforward alignment procedure and allows sharing optics with the fluorescent emission path. Our system employs an infrared (IR) laser that is passed through a double-hole mask to achieve two parallel beams which are made to reflect on the coverslip and subsequently detected on an industrial sCMOS camera. The relative position of these beams is then uniquely linked to the z-position of a microscope-mounted sample. The system was benchmarked by introducing temperature perturbations, where it was shown to achieve a stable focus, and by scanning different positions while simulating a perturbation in the z-position of the stage, where we show that a lost focus can be recovered within seconds.

biochemistry↗

bioRxiv2020

Actuation Enhances Patterning in Human Neural Tube Organoids

Tissues achieve their complex spatial organization through an interplay between gene regulatory networks, cell-cell communication, and physical interactions mediated by mechanical forces. Current strategies to generate in-vitro tissues have largely failed to implement such active, dynamically coordinated mechanical manipulations, relying instead on extracellular matrices which respond to, rather than impose mechanical forces. Here we develop devices that enable the actuation of organoids. We show that active mechanical forces increase growth and lead to enhanced patterning in an organoid model of the neural tube derived from single human pluripotent stem cells (hPSC). Using a combination of single-cell transcriptomics and immunohistochemistry, we demonstrate that organoid mechanoregulation due to actuation operates in a temporally restricted competence window, and that organoid response to stretch is mediated extracellularly by matrix stiffness and intracellularly by cytoskeleton contractility and planar cell polarity. Exerting active mechanical forces on organoids using the approaches developed here is widely applicable and should enable the generation of more reproducible, programmable organoid shape, identity and patterns, opening avenues for the use of these tools in regenerative medicine and disease modelling applications.

bioengineering↗

bioRxiv2020

Ultra-Low Colcemid Doses Induce Microtubule Dysfunction as Revealed by Super-Resolution Microscopy

Microtubule-interacting drugs, sometimes referred to as antimitotics, are used in cancer therapy to target and disrupt micro-tubules. However, their side effects require the development of safer drug regimens that still retain clinical efficacy. Currently, many questions remain regarding microtubule-interacting drugs at clinically relevant and ultra-low doses. Here, we use super-resolution microscopies (single molecule localization and optical fluctuation based) to reveal the initial microtubule dysfunctions caused by nanomolar concentrations of colcemid. Short exposure to 30 - 80 nM colcemid results in aberrant microtubule curvature while microtubule fragmentation is detected upon treatment with [≥]100 nM colcemid. Remarkably, even ultra-low doses (5 hours at <20 nM) led to subtle but significant microtubule architecture remodeling and suppression of microtubule dynamics. These challenges to microtubule function represent less severe precursor perturbations compared to the established antimitotic effects of microtubule-interacting drugs, and therefore offer potential for improved understanding and design of anti-cancer agents.

cell biology↗

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↗